Mutations of voltage-gated ion channnels that allow them to express a voltage-independent phenotype and an improved method to use the same

ABSTRACT

The subject invention includes mutant voltage-gated ion channels that are open over a wide range of potential differences across membranes. The present invention also includes methods of use such mutant voltage-gated ion channels in cells with highly negative potential differences across their membranes. One preferred mutant voltage-gated ion channel is a channel with a mutation at the residue homologous to P513 in Kv1.5 and at least one mutation at one of the residues homologous to R400, R403, and R409 in Kv1.5.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/395,272, filed Jul. 12, 2002, which is incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

This invention relates to mutated voltage-gated ion channels and theirability to complement cells deficient in uptake of the associated ionand to an improved method where the mutated ion channels function as adetection system for detecting inhibitors and/or activators of normallyvoltage-gated ion channels.

BACKGROUND

Voltage-gated ion channels control the permeability of cell membranes tospecific ions by opening and closing in response to changes in thepotential difference across the membrane. Many voltage-gated ionchannels play an essential role in information transfer and synapticfunctions in neurons. Voltage-gated ion channels also participate inneuronal integration, cardiac pacemaking, muscle contraction, hormonesecretion, cell volume regulation, lymphocyte differentiation, and cellproliferation. The ability to modulate the activity of voltage-gated ionchannels, neuronal and otherwise, has implications in a wide range ofphysiological systems, diseases, and conditions including, withoutlimitation, cardiac rhythm disturbances, diabetes, hypertension, asthma,and seizure disorders. As such, there is a need for methods ofidentifying compounds that can modulate the activity of voltage-gatedion channels. With current molecular biology techniques, voltage-gatedion channels may be expressed in a wide range of cells. The transgeniccells may be screened with libraries of compounds to identify compoundsthat modulate the activity. U.S. Pat. No. 5,795,770 discloses a methodfor screening yeast cells deficient in the uptake of potassium. However,most cells suitable for such screening have negative potentialdifferences across the cell membranes that keep the voltage-gated ionchannels closed. The problem is particularly acute with yeast, whichwould otherwise be a useful cell. The potential difference across theyeast cell membrane has been estimated at between −150 and −200 mV. Thetrk1 trk2 mutant disclosed in the U.S. Pat. No. 5,795,770 is even morehyperpolarized. Thus there is a need for mutations within conservedregions of voltage-gated ion channels that generate channels that areopen at highly negative potential differences for screening in cellswith highly negative potential differences across their cell membranessuch as yeast.

Voltage-gated ion channels (potassium, sodium, and calcium channels)assemble as homotetramers (MacKinnon, 1991) and in some casesheterotetramers with each of the 4 subunits composed of 6 transmembranehelices. The S5 and 56 transmembrane helices in each subunit, and theinterconnecting loops form the conduction pathway of the channels, alsoknown as the pore ((MacKinnon, 1991; Doyle et al., 1998). The S1 throughS3 helices do not have a dearly defined function yet, but likely areimportant determinants of channel structure and gating and thus arepotential targets for mutations that alter gating (Seoh et al., 1996).The helices which are the preferred targets for mutations of theinvention are the S4 helix, thought to be the voltage sensor (Noda etal., 1984; Papazian et al., 1991; Liman et al., 1991; Logothetis et al.,1992; Durell & Guy, 1992), and S6, which contributes to the pore of thechannel.

The S4 helix is composed of a regular array of up to seven positivelycharged amino acids (arginine or lysine) with 2 intervening hydrophobicresidues between each. The number, location, and character of theseresidues varies quite significantly in different channels and helpsdetermine their different responses to changes in transmembrane voltage.As these charged residues are located within the transmembrane electricfield, they should be influenced by changes in the voltage creating thisfield. The response of this positively charged helix to voltage changesis thought to lead to channel opening and this response can be measuredelectrophysiologically through gating currents and ionic currents whileresidue movement can also be directly measured using fluorescence. Thesemeasurements have led to many kinetic and biochemical models to explainthe process of opening of ion channels ((Zagotta et al., 1994; Bezanillaet al., 1994; Schoppa & Sigworth, 1998; Baker et al., 1998).

Partly because the breadth of the transmembrane region where the S4 islocated is unknown and the 7 charged S4 residues cover a large portionof the helix, it is thought that the residues will contribute varyingamounts of charge to the total gating charge. This has been supported bymutational studies which look at total gating charge per channelfollowing neutralization mutations of individual charged residues(Papazian et al., 1991; Perozo et al., 1994). In Shaker channels,neutralization of any one of the first 4 charged residues closest to theextracellular space (R362, R365, R368 and R371) shows significantreductions in total gating charge per channel. These 4 residues all seemto contribute to the total gating charge which moves upondepolarization. In Shaker channels there are an additional 3 chargedresidues (K374, R377 and K380) which are located closer to theintracellular space. Neutralization of K374 to glutamine results in nofunctional channel expression (Papazian et al., 1991; Perozo et al.,1994; Papazian et al., 1995; Aggarwal & MacKinnon, 1996); however,neutralization to a serine does permit expression and there is areduction in total gating charge, though smaller than the reductionobserved for the first 4 residues (Aggarwal & MacKinnon, 1996). Seoh etal. (Seoh et al., 1996) found no reduction in total gating charge whenK374Q was paired with the mutation E293Q which restores expression. The6th charged residue, R377 also shows no expression when mutated to theneutral glutamine (Papazian et al., 1995). However, histidine scanningmutagenesis suggests that this residue does not participate in gating(Bezanilla, 2000). Neutralization of the 7th residue, K380, does notlead to a reduction in gating charge (Papazian et al., 1991; Logothetiset al., 1992). It is difficult to draw any general conclusions fordifferent channels regarding the effects of these neutralizationmutations due to caveats regarding channel structure, but generally itseems that charged residues closer to the extracellular space (R362through R371) contribute more to total gating charge than residuescloser to the intracellular space (K374 through K380). Starkus, Raynerand associates ((Bao et al., 1999) have shown that the combined mutationof residues R362, R365 and R371 eliminates the voltage-sensitivity ofchannel gating in Shaker. Shaker channels harboring this combinationhave an opening probability of about 0.15 at all voltages between −160and +80 mV.

Based on the crystal structure of the Streptomyces lividans KcsA channel((Doyle et al., 1998), a tetramer of the N-terminal portion of the S6transmembrane domain forms the inner mouth of the pore of potassiumchannels like Shaker and Kv1.5. Mutation of one residue in Shaker, P475,to alanine has been reported to prevent channel opening. Mutation ofthis residue in Shaker to aspartate or glutamate stabilizes the channelin an open state (Hackos and Swartz, Biophys. J. 78:398A, Abstract 2349;PCT/US01/03963). In the related potassium channel, hKv1.5, we have foundas disclosed in this specification that neither combined mutation of theR362, R365 and R371 homologues nor mutation of the P475 are sufficientto rescue trk1 trk2 yeast, presumably owing to the hyperpolarizedmembrane.

SUMMARY OF THE INVENTION

The present invention concerns modified voltage-gated ion channels thatare open over a very wide potential difference range sufficient to allowscreening in organisms such as yeast that have a highly negativepotential difference across their membrane. The preferred embodiment ofthe present invention includes modification of a voltage-dependent ionchannel in the S4 and S6 regions that allows the channel to befunctional and pass ions effectively over a very wide potential rangebetween −200 mV and +100 mV and beyond. Such modifications are mostsimply made by modification of a nucleic acid sequence encoding thechannel and expressing the channel in a suitable cell type. A preferredembodiment is demonstrated in the Examples herein with the Kv1.5channel, a voltage-gated potassium channel. The invention includes othervoltage gated potassium, sodium and calcium ion channels as targets forscreening in organisms with highly negative potential differences acrosstheir membrane. The preferred embodiment Kv1.5QPD may be replicated inthese other voltage gated ion channels by mutation of homologousresidues. Such channels would include, for example, all voltage-gatedpotassium channels (Kv), and others within the HGNC gene family such ashERG and KCNQ channels. In addition, the mutations are also relevant tochannels that conduct other ions such as those within the Nav and Cavchannels gene families. All these channels have readily definable S4 andS6 regions and identifiable homologous amino acid residues to thosesuggested here that embody the invention.

The present invention further provides a process for detectingmodulators of voltage-dependent ion channels which comprises:

1. an improved method for treating modified cells with a test substance,wherein the modified cerevisiae cells express a nucleic acid sequencefor the modified voltage-dependent ion channel, a preferred embodimentof which are yeast cells;

2. robust growth of modified Saccharomyces cerevisiae cells expressingthe defined mutant channels(s) at very low concentrations of potassiumions (<0.5 mM K+), under which conditions untransformed trk1 trk2 yeastare unable to grow;

3. detecting any change in growth of the cells after treatment with thetest substance.

One aspect of the present invention is potassium channel comprising avoltage-gated potassium channel which when expressed in a mutant yeastdeficient in potassium uptake allows the mutant yeast to grow in thepresence of media with very low potassium concentration, wherein saidvoltage-gated potassium channel comprises one or more mutations whichproduces a constitutively open voltage-gated potassium channel. In anembodiment of the present invention, the very low potassiumconcentration is about 2 mM or less, about 1 mM or less, about 0.7 mM orless, about 0.5 mM or less, or about 0.2 mM or less. In anotherembodiment of the invention, the mutant yeast lacks TRK1 or TRK1 andTRK2 potassium transporter activity. In still another embodiment, themutations in the voltage-gated potassium channel are homologous toR400Q, and P513D in Kv1.5; are homologous to R403Q, and P513D in Kv1.5;are homologous to R409Q, and P513D in Kv1.5; are homologous to R400Q,R403Q, and P513D in Kv1.5; are homologous to R400Q, R409Q, and P513D inKv1.5; are homologous to R403Q, R409Q, and P513D in Kv1.5; or arehomologous to R400Q, R403Q, R409Q, and P513D in Kv1.5. In oneembodiment, the voltage-gated potassium channel is a member of an ionchannel family comprising Kv10, Kv11, Kv12, Kv1, Kv2, Kv3, or Kv4. Instill another embodiment, the voltage-gated potassium channel is Kv1.5or hERG.

Another aspect of the present invention includes a yeast cell comprisinga deficiency in potassium uptake and a constitutively open voltage-gatedpotassium channel which allows said yeast cell to grow in the presenceof media with very low potassium. In one embodiment, the deficiency isdue to a lack of TRK1 or TRK1 and TRK 2 potassium transporter activity.In another embodiment, the very low potassium concentration is about 2mM or less. In still another embodiment, the constitutively openvoltage-gated potassium channel comprises two or more mutations that arehomologous to R400Q, R403Q, R409Q, or P513D in Kv1.5. In yet anotherembodiment, the constitutively open voltage-gated potassium channelcomprises mutations homologous to R400Q, and P513D in Kv1.5; homologousto R403Q, and P513D in Kv1.5; homologous to R409Q, and P513D in Kv1.5;homologous to R400Q, R403Q, and P513D in Kv1.5; homologous to R400Q,R409Q, and P513D in Kv1.5; homologous to R403Q, R409Q, and P513D inKv1.5; or homologous to R400Q, R403Q, R409Q, and P513D in Kv1.5. Inanother embodiment, the constitutively open voltage-gated potassiumchannel is a member of an ion channel family comprising Kv10, Kv11,Kv12, Kv1, Kv2, Kv3, or Kv4. In an embodiment, the constitutively openvoltage-gated potassium channel is Kv1.5 or hERG.

Another aspect of the present invention includes a recombinant nucleicacid molecule comprising a nucleic acid sequence encoding any of theabove variations of the mutant voltage potassium channel disclosed aboveor in the specification. Another embodiment includes a promoter sequenceoperably linked such nucleic acid.

Still another aspect of the present invention includes methodscomprising providing a cell deficient in potassium uptake expressing thepotassium channels disclosed above, wherein said cell has a highnegative potential across the plasma membrane; growing the cell in verylow potassium; adding a compound; and assaying the effect of thecompound on the growth of the cell. In one embodiment, the cell is ayeast cell. In another embodiment, the yeast cell lacks TRK1 or TRK1 andTRK2 transporter activity. In still another embodiment, the yeast cellis S. cerevisiea. In yet anther embodiment, the effect of the compoundon the growth of the cell represents a procedure to determine themodulating activity of said compound on the potassium channel. Incertain embodiments, the modulating activity refers to the inhibitionactivity of said compound on the potassium channel.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Neither the Kv1.5 triple S4 mutant (A) nor the Kv1.5 P513D (B)S6 mutant grows on low K+ media. Each row represents differenttransformants in three columns of yeast dilutions, 1×10⁻⁴, 1×10⁻⁵ and1×10⁻⁶/ml. The control was vector only, without channel in rows 1, 3 and5. The channel transformants are in rows 2, 4 and 6, labeled Q124- in(A) and P513D in (B). Only row 4 of panel B shows apparent growth, butthis growth proved to result from pseudorevertant activity. The same istrue of the papillae seen in some patches.

FIG. 2. Growth on agar plates of yeast strains containing the Kv1.5 S6,triple S4 mutant in low potassium media. Six of nine transformants grewwell on 7 mM K medium. Two of nine control transformants grew weakly.Control was vector only, without channel in. Above are three examples.Top and bottom are channels, showing good growth and middle set ofplaques are vector alone. Three columns of yeast dilutions, 1×10⁻⁴,1×10⁻⁵ and 1×10⁻⁶/ml. Heaviest growth seen at 1×10⁻⁴ yeast/ml (rightcolumn). The left column only shows one colony in the top row, and isinvisible in the lower two rows.

FIG. 3. A graph of the actions of various Kv1.5 antagonists on thegrowth of Kv1.5 S6, triple 54 mutants in low potassium media. Yeastgrowth response for 6 Kv1.5 antagonists of differing potencies. The rankorder for inhibition of yeast growth is from Compound 1 to Compound 6.This order is very close to that obtained from the patch clamp assay.Individual data points are shown for two separate assays, and mean dataas the solid line.

FIG. 4. Alignment of select voltage-gated ion channels.

FIG. 5. Comparison of (A) hERG D540 vs. Kv1.5QPD and (B) hERG D540 vsWΔ3. (A) shows growth on 100 mM K+ (left plate) and growth on 0.5 mM KB(right plate). Kv1.5QPD (left side of each plate) allows growth at bothpotassium concentrations. hERG D540 (right side of each plate) does notsupport growth at 0.5 mM K+. (B) shows that hERG D540 (right side ofplate) allows growth on 5 mM K+ while WΔ3 alone (left side of plate)cannot grow on 5 mM K+.

BRIEF DESCRIPTION OF THE TABLES

Table 1. Inhibition of growth in the presence of the specific potassiumchannel-inhibiting agent, 4-aminopyridine.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The following definitions apply to terms used throughout thisspecification unless otherwise limited in specific instances.

The term ‘modified’ as used with respect to a cell, refers to a cell inwhich the wild-type genome has been altered by the addition of one ormore heterologous genes, a deficiency in one or more wild-type genes, ora combination thereof. Such modifications may be carried out bytransformation and homologous recombination through techniques wellunderstood by those having ordinary skill in the art.

The term ‘functional derivative or mutant thereof’ as used with respectto a protein refers to a protein differing from the subject protein byone or more amino acid residues but still having the ion channeltransport function of the protein and greater than about 90% sequencehomology. Such derivatives and mutants may include proteins that differfrom the wild-type protein by amino acid substitutions, deletions,disruptions, and the like. Such differences may be accomplished bygenetic means, using such techniques as site-directed mutagenesis, ormutagenic PCR prior to translation, or by chemical means using proteasesand/or ligases after translation.

The functional derivatives of human or mammalian ion channel genesincorporate specific mutations into the voltage-sensing regions of theS4 transmembrane domain and in the pore-lining surface of S6 in eachinstance. Specifically three of the outer positively charged arginine orlysine residues of the S4 domain are mutated to glutamine residues and aconserved proline in the S6 domain is mutated to aspartate. This altersthe functional properties of channel opening, also known as gating.Potassium ion channels normally only gate open when the perceivedpotential across the protein (usually incorporated into a lipid bilayer)exceeds, or becomes positive to −40 mV on the inside. With the presentmodification the channels gate across an extremely wide potential rangeexceeding −200 mV in the negative direction and +100 mV in the positivedirection. This allows them to be open at transmembrane potentials atwhich they are normally dosed (i.e. negative to −60 mV on the inside).

The term “channel ion”, as used herein, refers to the ion which a givenvoltage-gated ion channel selectively passes when open. Thevoltage-gated ion channels of the present invention may be potassium,sodium, or calcium channels.

As used herein, the term “wide-potential-range ion channel” refers to amutant voltage-gated ion channel that is open to a sufficient degree athighly negative potential differences to complement a yeast deficient inuptake of the channel ion. A preferred embodiment ofwide-potential-range ion channels is one that can allow the yeastdeficient in uptake of the channel ion when the channel ion is suppliedat a low concentration in the media. Thus a wide-potential-range ionchannel may allow yeast deficient in uptake of the channel ion when thechannel ion is less than 2 mM, less than 1.0 mM, less than 0.5 mM orless than 0.2 mM. The Examples provide a preferred embodiment of awide-range-potential ion channel in the form of Kv1.5QPD.

As used herein, the term “highly-negative potential difference” means apotential difference across a membrane of −200 mV or greater. In someembodiments, the highly-negative potential difference is −225 mV orgreater, −250 mV or greater, or −300 mV or greater. In a preferredembodiment, the potential difference is that found in trk1 trk2 yeastgrown in media with 0.5 mM potassium.

Voltage-Gated Ion Channels

Voltage-gated ion channels that are mutated in the present invention arefrom related families of proteins. Voltage-gated ion channels may bereadily identified by function, by structure (both secondary andtertiary), and by sequence homology (primary structure). A hallmark ofthe voltage-gated ion channels are the six putative transmembranespanning helices S1-6 and the “PVP” motif (which is not invariant, e.g.,rKv2.1 has a PIP sequence). Within the larger super-family are variousfamilies including potassium gated (Kv), sodium gated (Nav), and calciumgated (Cav). The voltage-gated potassium channels fall into asuper-family that uses the nomenclature Kv. One family includes foursub-families that were originally named for the four related voltagegated potassium channels from Drosophila: Shaker (Kv1); Shab (Kv2); Shaw(Kv3); and Shal (Kv4). Shaker and Shal are characterized as having rapidcurrent activation and inactivation, while Shab and Shaw are delayedrectifier channels that are characterized as having slow inactivationand non-inactivation. Homologues in each subfamily have been identifiedin humans, rodents, and other mammals. FIG. 4 shows an alignment ofrepresentative members of the Kv1-K4 sub-families.

In addition, the Kv super-family has a number of other families.Kv10-Kv12 make up the eagrelated family. Human erg (Herg or Kv11.1) isan important member of this family. The Herg gene corresponds to theLQT-2 genetic locus. Long QT (LQT) syndromes are inherited cardiacdisorders characterized by prolonged QT interval on theelectrocardiogram (ECG) associated with syncopal attacks, and high riskof sudden death due to ventricular tachyarrhythmia (Schwartz, 1985).Although congenital LQTs are not frequent diagnoses, acquired forms ofabnormal repolarization and susceptibility to arrhythmia are verycommon. Occasional syncopes (loss of consciousness) are due to malignanttacharyrhythmias, usually torsade de pointes. Sudden death may occur bytransformation of these ventricular arrhythmias into ventricularfibrillation. Mutations in Herg are known to cause autosomal dominantLQT syndrome. Herg ion channels are inwardly rectifying potassiumchannels that have voltage gating properties similar to other members ofother eag-related channels. The most common form of LQTs, however, isdue to mutations in the KvLQT1 (Kv7.1) potassium channel gene thatencodes an outwardly rectifying, voltage-gated K+ channel with sixtransmembrane domains.

The present invention provides mutant voltage-gated ion channels thatremain open at extremely negative potential differences. The mutantvoltage-gated ion channels of the present invention are characterized bytheir ability to complement yeast deficient in potassium uptake evenwhen grown at very low potassium levels. The preferred levels are 0.5 mMK⁺ or lower. A preferred embodiment of the present invention is achannel that includes mutations at residues homologous to R400, R403,R409, and/or P513 in Kv1.5. A further preferred embodiment of thepresent invention is a channel that includes mutations homologous toR400Q, R403Q, R409Q, and/or P513D in Kv1.5.

The mutant voltage-gated ion channels of the present invention may begenerated by a wide range of well known molecular biology techniques.Detailed protocols for numerous such procedures are described in, e.g.,in Ausubel et al. Current Protocols in Molecular Biology (Supplementedthrough 2000) John Wiley & Sons, New York; Sambrook et al. MolecularCloning—A Laboratory Manual (2^(nd) Ed.), Vol. 1-3, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 1989, and Berger and Kimmel Guideto Molecular Cloning Techniques, Methods in Enzymology volume 152Academic Press, Inc., San Diego, Calif.

Transgenic Cells of the Present Invention

The present invention also encompasses transgenic cells expressing themutant voltage-gated ion channels. A preferred cell is one withhighly-negative potential differences across its cell membrane, such asyeast. One of ordinary skill in the art will be able to generate thetransgenic cells of the present invention using routine methods ofmolecular biology. The transgene may be constructed within or introducedinto an expression vector appropriate for the cell of interest. Manydifferent expression vectors exist for expression in a variety ofprokaryotic and eukaryotic cells. For ease of manipulation, a shuttlevector may be used to allow manipulation of the construct in aprokaryotic cell and later expression in a eukaryotic cell.

Such expression vectors typically contain a promoter and a transcriptiontermination element. The transgene should be operably linked to apromoter that functions in the cell of interest. The promoter may beconstitutive or inducible. The choice of promoter will be dictated bythe particular application. One of skill in the art would have nodifficulty in selecting the appropriate promoter. Optionally, theexpression vector may also contain other useful elements such asribosome binding sites, polyadenylation signals, etc. as appropriate.

Once the expression vector has been constructed, it may be introducedinto the cell of interest by a number of standard transfection methods.These may include calcium phosphate transfection, protoplast fusion,electroporation, microprojectile bombardment, liposomes, microinjection,viral vectors and any other known method for introducing cloned nucleicacid sequences into the cell of interest as is appropriate. All thatmatters is that the nucleic acid is introduced into the cell ofinterest. The expression vector may integrate into the genetic materialof the host cell, may replicate as an independent genetic element, ormay remain in the cell transiently.

Once the expression vector has been introduced into the cell. The cellmay be cultured under conditions that allow expression of the mutantvoltage-gated ion channel when desired.

Screening Methods of the Present Invention

The present invention also provides methods for screening compounds fortheir ability to modulate the activity of voltage-gated ion channels.The mutant voltage-gated ion channels of the present invention may beexpressed in a cell with a highly negative potential difference acrossthe membrane. The cell should be deficient in uptake of the channel ionof the voltage-gated ion channel of interest such that growth isinhibited or hindered on media containing low concentrations of thechannel ion of the mutant voltage-gate ion channel. Such a cell-basedsystem may be assayed to determine whether a given compound modulatesthe activity of the voltage-gated ion channel. The modulation may bydetected by the effect on the growth of the cell or other methods thatdetect the presence of the channel ion within the cell such as use ofcompounds that fluoresce in the presence of the channel ion.

Cells deficient in the uptake of the channel ion of the voltage-gatedion channel of interest may be generated by many different techniquesavailable to those of skill in the art. The cell may be made deficientby targeted knock-out of genes involved in conveying the channel ionacross the membrane. One of skill in the art will recognize that thismay be accomplished in several ways, including knock-out of the genesfor the proteins that actually convey the channel ions across themembrane as channels or transporters; knock-out of the genes thatactivate the transcription of such genes; etc. In addition, the cell maybe made by screening for naturally occurring mutations or mutationsinduced by a variety of methods such as chemical mutagenesis, transposoninsertion, radiation, etc. Furthermore, the cell may be made deficientby addition of compounds that inhibit the cell's natural mechanisms foruptake of the channel ion. The method used to make the cell deficient isnot important, so long as the cell is rendered deficient such that thecell cannot grow in media with low concentrations of the channel ion.

The compounds tested for the ability to modulate the activity ofvoltage-gated ion channels can include any small chemical entity as wellas biological entities such as proteins, sugars, nucleic acids, lipids,and any combination thereof. It will be appreciated that the screeningmethod of the present invention may be applied to large numbers ofcompounds. In a preferred embodiment, the assay is part of an automatedhigh-throughput screening apparatus designed to screen large librariesof chemical and/or biological entities.

The compounds identified may be used to modulate the activity of thevoltage-gated ion channels in vivo by administering the compound to ananimal that expresses the voltage-gated ion channel. It will beappreciated by those of ordinary skill in the art that such compoundswill be defined by their function in the screening assays of the presentinvention as well as structurally for those that modulate by directinteraction with the voltage-gate ion channel. The structure of such acompound must at least in part be defined by the structure of thevoltage-gated ion channel much as the structure of an electrical plug isin part defined by an electrical socket. Thus, the compounds identifiedin the assays of the present invention and their methods of use tomodulate the voltage-gated ion channels in animals is an aspect of thepresent invention.

A preferred embodiment of the present invention is yeast cell deficientin potassium uptake for use in screening voltage-gated potassiumchannel. The yeast cell of the present invention possesses deficientalleles in the TRK1 and TRK2 genes required for potassium uptake. Themodified alleles are genetically stable and recessive, so they can becomplemented with activities encoded by heterologous genes introducedinto the strain (U.S. Pat. No. 5,795,770). Pseudorevertants do arisewith high frequency, however, and can complicate the strain's usefulnessin liquid culture. The present invention provides a method to overcomethis problem in liquid culture.

A variety of the modified voltage-gated ion channels containing thecombined mutations in homologous positions to those described above maybe introduced into this yeast strain to assess whether these channelscan complement the growth defect on potassium-deficient media. Theapplication results in a yeast strain expressing a normallyvoltage-gated, modified foreign ion channel, useful in a screen formodulators of the channels.

A yeast strain expressing an altered voltage-gated ion channel can beadapted to natural products screening. A simple screen design involvinggrowth inhibition or ion uptake on solid plates or in liquid culture maydetect compounds that modulate channel function. The screen may involvesuch modifications as sensitivity to the pH of the media, growth indifferent concentrations of oxygen and carbon dioxide, changes in growthon different concentrations of potassium in the medium, or changes inion uptake.

EXAMPLES Example 1 Construction of Mutant Kv1.5 Channels

The gene for human potassium channel Kv1.5 was cloned into yeastexpression vectors such that expression was under the control of theGALL or PGK promoter. Site-directed mutagenesis was then used to recodethe Kv1.5 gene such that amino acids R400, R403, and R409 would bereplaced by glutamine and amino acid P513 would be replaced by aspartatein the expressed channel. The coding region of the human gene for Kv1.5was subcloned into pYC2 (Invitrogen) as an approximately 2 kbHindIII-NotI fragment. Site-directed mutagenesis was conducted in twosteps using the Quick Change Mutagenesis Kit (Invitrogen). Theoligonucleotides 5′-ATCCTCCAAGTCATCCAACTGGTCCGGGTGTTCCAAATCTTCAAG-3′(SEQ ID NO: 1) and 5′-TTGMGATTGGAACACCCGGACCAGTrGGATGACrGGAGGATG-3′(SEQID NO: 2) encoded the five nucleotide changes in the S4-coding regionfrom wild-type. The oligonucleotides5′-ATTGCCCTGCCTGTGGACGTCATCGTCTCCAAC-3′ (SEQ ID NO: 3) and5′-TTGGAGACGATGACGTCCACAGGCAGGGCAATG-3′ (SEQ ID NO: 4) encoded the 2nucleotide changes in the S6 coding region. The presence of thepredicted nucleotide changes was confirmed by DNA sequencing. Furthersequencing confirmed the absence of additional mutations.

The replacement of the three arginines was based on the fact that in arelated channel, Shaker, the analogous replacements yield a channelwhich gates in a voltage-insensitive manner ((Bao et al., 1999). Theproline at position 513 was replaced on the basis of a report that ananalogous mutation in Shaker yielded a constitutively open channel(Hackos and Swartz, Biophys. J. 78:398A, Abstract 2349). In Kv1.5,neither the changes in S4 nor those in S6 proved sufficient on their ownto allow yeast growth in the screen described below (FIG. 1).

The plasmid containing the mutant Kv1.5 channel (Kv1.5m) of the presentinvention was transformed into the trk1trk2 yeast strain using a Lithiumacetate procedure. Unlike the trk1 trk2 yeast transformed with the emptyvector, the quadruple-mutant Kv1.5m construct expressed from the GAL1promoter (with galactose used as the carbon source) when transformedinto these yeast, was found to allow growth on media containing 5 mMpotassium (growth on glucose, as expected, required 100 mM potassium.Kv1.5m is under the control of the GAL1 promoter, and, thus, should beexpressed only when galactose is the sole carbon source). A similarphenomenon was observed in yeast transformed with a vector expressingKv1.5m from the constitutive PGK promoter, but independent of carbonsource. Neither the combined three arginine mutations nor the prolinemutation on their own conferred growth capability on low potassium tothe yeast (see above, FIG. 1). The Kv1.5m-transformed strain is apreferred embodiment of this invention.

Growth of the trk1 trk2 strain expressing Kv1.5m on low potassium couldbe blocked by the addition of the Kv1.5-blocking 4-aminopyridine (Table1), as well as by other agents known to block Kv1.5 (FIG. 3). Growth ofthe trk1trk2 strain transformed with a similar construct expressing theinward rectifier KIR2.1 rather than Kv1.5m was not inhibited by thesedrugs.

The trk1 trk2 mutant yeast, WΔ3 (MATa, leu2-3,112, trp1-1, ura3-3,ade2-1, his3-1, can1-100, trk1::LEU2, trk2::HIS3), was a gift of AlonsoRodriguez-Navarro, Escuela Tecnica Superior de Ingenieros Agronomos,Madrid, Spain.

Example 2 Introduction of the Ion Channel Mutant Forms into the Yeast

YPD, YNB and low-salt (LS) media were prepared by standard methods(Sherman, Fink and Hicks, Methods in Yeast Genetics, Cold Spring Harbor,1986; Rodriguez-Navarro and Romos, J. Bacteriol. 159:940-945,1984;Wickerham, L. J., U.S. Dept of Agriculture Technical Bulletin No. 1029,1951). Introduction of the modified gene incorporated in expressionvectors into yeast was by a simple transformation procedure. Yeast cellswere incubated overnight at room temperature with the plasmid DNA andcarrier (salmon sperm DNA) in 100 mM Lithium acetate, 10 mM Tris HCl, 1mM EDTA, 34% polyethylene glycol (m.w. 4000). Initial selection andsubsequent screening of transformants was carried out on media lackinguracil (YNB-uracil/100 mM KCl) to maintain selection for the plasmids.100 mM KCl was included to allow time for Kv1.5m expression. The parentstrain, WΔ3, is capable of growth on 100 mM KCl but not on 7 mM KCl orlower. Transformant colonies were then transferred to low-potassium (7mM) media lacking uracil (YNB-uracil) to verify functional expression ofthe channel as shown in FIG. 2.

Example 3 Function of the Cells as Reporters in Drug Screening

Expression of the cloned inserts is under the control of either theconstitutively active PGK promoter or the inducible GAL1 promoter. Thelatter requires growth in/on media containing galactose as sole carbonsource. The screening method involves adding the compound to be screenedto the growth media of trk1 trk2 yeast expressing the modified potassiumchannel and determining whether the yeast's growth is inhibited. Thetested compound may be incorporated in liquid or solid growth media oradded ectopically to solid growth media.

A major problem in using trk1 trk2 yeast is the high number ofpseudorevertants that generally arise on low potassium media (Liang etal., 1998). This is not a significant problem on solid media, since thepseudorevertants appear as individual colonies on a non-growingbackground (Graves & Tinker, 2000). But in liquid cultures, thefrequency is high enough that virtually all overnight cultures areovertaken by the pseudorevertants. This problem is endemic in culturesgrown in 7 mM potassium as described by all other workers. Sinceinsensitive pseudorevertants mask any block of the expressed channel,drug sensitivity testing in liquid culture is nearly impossible underthese conditions

In the present invention a surprisingly simple method was found by theinventors to overcome this problem. Our yeast strain, when expressingthe mutant Kv1.5 channel, is capable of consistent growth on as littleas 0.5 mM potassium. Very few pseudorevertants are capable of growth atthis potassium concentration; the vast majority of liquid cultures ofthe parent strain show absolutely no growth even after more than 24hours culture. When expressing the mutant Kv1.5 channel, growth isrobust and can be blocked by known Kv1.5 channel blockers.

Detailed Description of the Methods Required for a Liquid Culture MediaAssay of Kv1.5:

Low salt media was made according to Wickerham, L. J. (1951) (Taxonomyof Yeasts. United States Department of Agriculture Technical BulletinNo. 1029), except that the final potassium phosphate concentration was0.5 mM.

1. Streak out WΔ3 and Kv1.5m transformed WΔ3 (“Kv1.5m”) culture fromfrozen stock onto YPD+100 mM K+ and YNB+100 mM K+, ura-agar mediaplates, respectively, for fresh colonies.

2. The day before doing the drug assay, use the fresh colonies to growovernight cultures of WΔ3 in liquid YPD+100 mM K+ media, and grow “Kv1.5m” in liquid YNB+100 mM K+, ura-media.

3. Collect the overnight yeast cells and wash them in sterile distilledwater. Resuspend WΔ3 cells in the same volume of LS+100 mM K++ uracilmedia, and resuspend “Kv1.5 m” cells in the same volume of LS+0.5 mM K+,ura-media. These are the seed cultures for the drug assay.

4. In Falcon 2059 tubes, prepare various concentrations of drugs inLS+0.5 mM K+, ura-media or LS+100 mM K++ uracil media, and also set upsolvent controls for each drug concentrations used. Here is an example:

Drug X stock solution (10 mM) is dissoved in 0.5% Tween-20. Finalconcentrations that we test are 1 mM, 0.1 mM, and 0.01 mM. WΔ3 controlset up: 0.1 mM 0.1 mM 0.01 mM 0 mM drug × (10 mM) 0.5 ml 0.05 ml 0.005ml — LS + 100 mM K+ + 4.5 ml 4.95 ml 4.995 ml 5 ml uracil media

Then 25 micro liter of the seed culture of WΔ3 from step 3 is inoculatedinto each tube. Solvent control set up: 0.1 mM 0.1 mM 0.01 mM 0 mM 0.5%Tween-20 0.5 ml 0.05 ml 0.005 ml — LS + 100 mM K+ + 4.5 ml 4.95 ml 4.995ml 5 ml uracil media

Then 25 micro liter of the seed culture of WΔ3 from step 3 is inoculatedinto each tube. Drug assay set up: 0.1 mM 0.1 mM 0.01 mM 0 mM Drug × (10mM) 0.5 ml 0.05 ml 0.005 ml — LS + 0.5 mM K+, 4.5 ml 4.95 ml 4.995 ml 5ml ura-media

Then 250 micro liter of the seed culture of “Kv1.5m” from step 3 isinoculated into each tube.

5. Measure the starting OD600 by taking out 0.1 ml of the sample fromeach tube and diluting lox for measuring. Grow cultures at 300 C shakerat 300 rpm.

6. Get 0.1 ml of samples from each tube and measure their OD600 at 2 hr,4 hr, 6 hr, 8 hr, and 24 hr.

Table 1 shows that the standard potassium channel blocker,4-aminopyridine (4-AP) can block the Kv1.5m channels in the liquid mediaassay: Inhibition of the Growth of QPD WΔ3 Transformants by 4-AP inLiquid Culture Media OD600 (Batch A) OD600 (Batch B) LS + 0.5 mM K⁺ 2.332.33 LS + 0.5 mM K⁺ + 0.1 mM 4-AP 2.55 2.5 LS + 0.5 mM K⁺ + 1 mM 4-AP1.11 1.11

The reduction of 50% at 1 mM 4-AP in both batches at OD600 indicates aninhibition of yeast growth by the presence of the compound.

In addition, this liquid assay reproducibly matches the rank order ofpotency of a number of Kv1.5 antagonists, as shown in FIG. 3. Thepotency (EC50) of the various compounds used on Kv1.5 as determined bypatch clamp metholodogy (n=3) determinations, was, in rank order ofpotency:

Compound 1 (Stock solution dissolved in 0.5% Tween 20 at 10 mM,EC50=0.29-0.43 micro molar)

Compound 2 (water soluble, EC50=0.4 micro molar)

Compound 3 (water soluble, EC50=3.6 micro molar)

Compound 4 (water soluble, EC50=5.4 micro molar)

Compound 5 (water soluble. EC50=6.1-10.2 micro molar)

Compound 6 (water soluble, EC50=28 micro molar)

Example 4 Test with Another Shaker Channel

A second shaker channel was tested to determine whether a previouslyreported single mutation would function to allow a trk1 trk2 yeast togrow at very low potassium concentration. hERG with a single mutation,D540K, has been reported to yield a channel that is open athyperpolarized potentials (Sanguinetti and Xu, 1999). These workersreported that the D540K mutation (in the channel's S4-S5 linker) causeshERG to open at hyperpolarized potentials into a novel long-lived openstate. This mutant may be used for screening at 5.0 mM K+. As shown inFIG. 5, however, this mutation is not sufficient to allow trk1 trk2yeast growth on 0.5 mM K+.

One of skill in the art will recognize, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

REFERENCES

All references cited hereunder are hereby incorporated by reference.

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1. A potassium channel comprising a voltage-gated potassium channelwhich when expressed in a mutant yeast deficient in potassium uptakeallows the mutant yeast to grow in the presence of media with very lowpotassium concentration, wherein said voltage-gated potassium channelcomprises one or more mutations which produces a constitutively openvoltage-gated potassium channel.
 2. The potassium channel of claim 1,wherein the very low potassium concentration is about 2 mM or less. 3.The potassium channel of claim 1, wherein the very low potassiumconcentration is about 1 mM or less.
 4. The potassium channel of claim1, wherein the very low potassium concentration is about 0.7 mM or less.5. The potassium channel of claim 1, wherein the very low potassiumconcentration is about 0.5 mM or less.
 6. The potassium channel of claim1, wherein the very low potassium concentration is about 0.2 mM or less.7. The potassium channel of claim 1, wherein the mutant yeast lacks TRK1or TRK1 and TRK2 potassium transporter activity.
 8. The potassiumchannel of claim 7, wherein the mutant yeast lacks TRK1 and TRK2potassium transporter activity.
 9. The potassium channel of claim 1,wherein the mutations in the voltage-gated potassium channel arehomologous to R400Q, and P513D in Kv1.5.
 10. The potassium channel ofclaim 1, wherein the mutations in the voltage-gated potassium channelare homologous to R403Q, and P513D in Kv1.5.
 11. The potassium channelof claim 1, wherein the mutations in the voltage-gated potassium channelare homologous to R409Q, and P513D in Kv1.5.
 12. The potassium channelof claim 1, wherein the mutations in the voltage-gated potassium channelare homologous to R400Q, R403Q, and P513D in Kv1.5.
 13. The potassiumchannel of claim 1, wherein the mutations in the voltage-gated potassiumchannel are homologous to R400Q, R409Q, and P513D in Kv1.5.
 14. Thepotassium channel of claim 1, wherein the mutations in the voltage-gatedpotassium channel are homologous to R403Q, R409Q, and P513D in Kv1.5.15. The potassium channel of claim 1, wherein the mutations in thevoltage-gated potassium channel are homologous to R400Q, R403Q, R409Q,and P513D in Kv1.5.
 16. The potassium channel of claim 1 or 7, whereinthe voltage-gated potassium channel is a member of an ion channel familycomprising Kv10, Kv11, and Kv12.
 17. The potassium channel of claim 1 or7, wherein the voltage-gated potassium channel is a member of the Kv10ion channel family.
 18. The potassium channel of claim 1 or 7, whereinthe voltage-gated potassium channel is a member of the Kv11 ion channelfamily.
 19. The potassium channel of claim 1 or 7, wherein thevoltage-gated potassium channel is a member of the Kv12 ion channelfamily.
 20. The potassium channel of claim 1 or 7, wherein thevoltage-gated potassium channel is a member of an ion channel familycomprising Kv1, Kv2, Kv3, and Kv4.
 21. The potassium channel of claim 1or 7, wherein the voltage-gated potassium channel is a member of the Kv1ion channel family.
 22. The potassium channel of claim 1 or 7, whereinthe voltage-gated potassium channel is a member of the Kv2 ion channelfamily.
 23. The potassium channel of claim 1 or 7, wherein thevoltage-gated potassium channel is a member of the Kv3 ion channelfamily.
 24. The potassium channel of claim 1 or 7, wherein thevoltage-gated potassium channel is a member of the Kv4 ion channelfamily.
 25. The potassium channel of claim 1 or 7, wherein thevoltage-gated potassium channel is Kv1.5 or hERG.
 26. The potassiumchannel of claim 1 or 7, wherein the voltage-gated potassium channel isKv1.5
 27. The potassium channel of claim 1 or 7, wherein thevoltage-gated potassium channel is hERG.
 28. A yeast cell comprising adeficiency in potassium uptake and a constitutively open voltage-gatedpotassium channel which allows said yeast cell to grow in the presenceof media with very low potassium.
 29. The yeast cell of claim 28,wherein the deficiency is due to a lack of TRK1 or TRK1 and TRK 2potassium transporter activity.
 30. The yeast cell of claim 28, whereinthe deficiency is due to a lack of TRK1 and TRK 2 potassium transporteractivity.
 31. The yeast cell of claim 28 or 29, wherein the very lowpotassium concentration is about 2 mM or less.
 32. The yeast cell ofclaim 28 or 29, wherein the constitutively open voltage-gated potassiumchannel comprises one or more mutations that are homologous to R400Q,R403Q, R409Q, or P513D in Kv1.5.
 33. The yeast cell of claim 28 or 29,wherein the constitutively open voltage-gated potassium channelcomprises two or more mutations that are homologous to R400Q, R403Q,R409Q, or P513D in Kv1.5.
 34. The yeast cell of claim 28 or 29, whereinthe constitutively open voltage-gated potassium channel comprisesmutations homologous to R400Q, and P513D in Kv1.5.
 35. The yeast cell ofclaim 28 or 29, wherein the constitutively open voltage-gated potassiumchannel comprises mutations homologous to R403Q, and P513D in Kv1.5. 36.The yeast cell of claim 28 or 29, wherein the constitutively openvoltage-gated potassium channel comprises mutations homologous to R409Q,and P513D in Kv1.5.
 37. The yeast cell of claim 28 or 29, wherein theconstitutively open voltage-gated potassium channel comprises mutationshomologous to R400Q, R403Q, and P513D in Kv1.5.
 38. The yeast cell ofclaim 28 or 29, wherein the constitutively open voltage-gated potassiumchannel comprises mutations homologous to R400Q, R409Q, and P513D inKv1.5.
 39. The yeast cell of claim 28 or 29, wherein the constitutivelyopen voltage-gated potassium channel comprises mutations homologous toR403Q, R409Q, and P513D in Kv1.5.
 40. The yeast cell of claim 28 or 29,wherein the constitutively open voltage-gated potassium channelcomprises mutations homologous to R400Q, R403Q, R409Q, and P513D InKv1.5.
 41. The yeast cell of claim 28 or 29, wherein the constitutivelyopen voltage-gated potassium channel is a member of an ion channelfamily comprising Kv10, Kv11, and Kv12.
 42. The yeast cell of claim 28or 29, wherein the constitutively open voltage-gated potassium channelis a member of the Kv10 ion channel family.
 43. The yeast cell of claim28 or 29, wherein the constitutively open voltage-gated potassiumchannel is a member of the Kv11 ion channel family.
 44. The yeast cellof claim 28 or 29, wherein the constitutively open voltage-gatedpotassium channel is a member of the Kv12 ion channel family.
 45. Theyeast cell of claim 28 or 29, wherein the constitutively openvoltage-gated potassium channel is a member of an ion channel familycomprising Kv1, Kv2, Kv3, and Kv4.
 46. The yeast cell of claim 28 or 29,wherein the constitutively open voltage-gated potassium channel is amember of the Kv1 ion channel family.
 47. The yeast cell of claim 28 or29, wherein the constitutively open voltage-gated potassium channel is amember of the Kv2 ion channel family.
 48. The yeast cell of claim 28 or29, wherein the constitutively open voltage-gated potassium channel is amember of the Kv3 ion channel family.
 49. The yeast cell of claim 28 or29, wherein the constitutively open voltage-gated potassium channel is amember of the Kv4 ion channel family.
 50. The yeast cell of claim 28 or29, wherein the constitutively open voltage-gated potassium channel isKv1.5 or hERG.
 51. The yeast cell of claim 28 or 29, wherein theconstitutively open voltage-gated potassium channel is Kv1.5.
 52. Theyeast cell of claim 28 or 29, wherein the constitutively openvoltage-gated potassium channel is hERG.
 53. A recombinant nucleic acidmolecule comprising a nucleic add sequence encoding the potassiumchannel of claim
 1. 54. A recombinant nucleic acid molecule comprising apromoter sequence operably linked to the nucleic acid molecule of claim53.
 55. A method comprising: (a) providing a cell deficient in potassiumuptake expressing the potassium channel of claim 1, wherein said cellhas a high negative potential across the plasma membrane; (b) growingthe cell in very low potassium; (c) adding a compound; and (d) assayingthe effect of the compound on the growth of the cell.
 56. The method ofclaim 55, wherein the cell is a yeast cell.
 57. The method of claim 56,wherein the yeast cell lacks TRK1 or TRK1 and TRK2 transporter activity.58. The method of claim 56, wherein the yeast cell lacks TRK1 and TRK2transporter activity.
 59. The method of claim 57, wherein the yeast cellis S. cerevisiea.
 60. The method of claim 55, wherein assaying theeffect of the compound on the growth of the cell represents a procedureto determine the modulating activity of said compound on the potassiumchannel of claim
 1. 61. The method of claim 60, wherein the modulatingactivity refers to the inhibition activity of said compound on thepotassium channel of claim 1.